G-protein coupled receptors (GPCRs) constitute the largest family of membrane receptors in the cell. They transduce extracellular signals to intracellular effector systems and are involved in a large variety of physiological phenomena, therefore representing the most common target of pharmaceutical drugs although only a small percentage of GPCRs are targeted by current therapies.
GPCRs respond to a wide range of ligands. Due to the progress of human genome sequencing, for about 25% out of the more than 400 GPCRs (not including the olfactory GPCRs) that have been identified, a defined physiologically relevant ligand is still lacking. These receptors are known as “orphan GPCRs”. “Deorphanization” and identification of their in vivo roles is expected to clarify novel regulatory mechanisms and, therefore, to disclose novel drug targets. Whether GPR17 is such an orphan receptor is still a matter of debate. Phylogenetically, GPR17 is most closely related to the nucleotide P2Y receptors and the cysteinylleukotriene (CysLT1, CysLT2) receptors, with an amino acid sequence identity of between about 30 and about 35%, respectively.
Multiple-tissue Northern blot and RT-PCR analyses indicate a predominant expression of GPR17 in the central nervous system (CNS) (Clana et al., 2006, EMBO J. 25(19): 4615; Blasius et al., 1998, J Neurochem 70(4): 1357) and additionally in heart and kidney, i.e. organs typically undergoing ischemic damage. Two GPR17 isoforms have been identified differing only by the length of their N-terminus. The short GPR17 isoform encodes a 339 amino acid—residue protein with typical rhodopsin type-seven transmembrane motifs. The long isoform encodes a receptor with a 28 amino acid longer N-terminus (Blasius et al., 1998). GPR17 is highly conserved among vertebrate species (˜90% identity of amino acid sequence to both mouse and rat orthologs), which may constitute an advantageous feature for development of small molecule ligands and animal models in a drug discovery context.
In the original deorphaning report, GPR17 was identified as dual receptor for uracil nucleotides and cysteinyl-leukotrienes (cysLTs) LTC4 and LTD4, respectively based on 35SGTPγS binding and cAMP inhibition assays as well as single cell calcium imaging (Clana et al., 2006). Evidence for GPR17 functionality was provided in different cellular backgrounds such as 1321N1, COS7, CHO, and HEK293 cells (Clana et al., 2006). Subsequently, an independent study confirmed activation of GPR17 by uracil nucleotides but failed to recapitulate activation by CysLTs (Benned-Jensen, 2010, Br J Pharmacol, 159(5): 1092). Yet another very recent report (Maekawa et al., 2009) suggested lack of GPR17 responsiveness to both uracil nucleotides and CysLTs across three different cellular backgrounds stably expressing GPR17 (1321N1, CHO, HEK293 cells). Instead a novel regulatory role for GPR17 was proposed: GPR17—upon coexpression with CysLT1—rendered CysLT1 unresponsive to its endogenous lipid mediators LTC4 and LTD4. Clearly, additional in vitro investigations are required to probe GPR17 pharmacology and function in more depth.
Drugs modulating the GPR 17 activity may have neuroprotective, anti-inflammatory and anti-ischemic effects and may thus be useful for the treatment of cerebral, cardiac and renal ischemia, and stroke (WO 2006/045476). WO 2005/103291 disclosed analgetic effects of a GPR 17 agonist and proposed the use of GPR 17 agonists for treating neuropathic pain. Moreover, evidence is accumulating that GPR 17 is involved in myelination processes and that GPR 17 antagonists can be valuable drugs for the treatment or alleviation of myelination disorders such as multiple sclerosis or spinal cord injury (Chen et al, Nature neuroscience 2009, 12(11):1398-406; Ceruti et al; Brain: a journal of neurology 2009 132(Pt 8):2206-18). The identification of potent and selective GPR 17 modulators could thus be of significant relevance in the treatment of these serious diseases.
Identification of an activating ligand is a prerequisite to search for compounds that modulate GPR17 activity. Although activation by uracil nucleotides and cysteinyl-leukotrienes of GPR17 has been reported (Clana et al., 2006; Pugliese et al., 2009, Am J Physiol Cell Physiol 297: C1028), these endogenous signalling molecules do not display functional activity in different cell lines (1321N1, CHO, HEK) engineered to stably express the short isoform of GPR17 in our laboratory. In agreement with our observations, inactivity of these ligand classes has also been observed in a recent study (Maekawa et al., 2009, PNAS, US, 106(28): 11685). Another independent laboratory was also not able to confirm cysteinyl-leukotrienes as GPR17 agonists (Benned-Jensen, 2010), while depending on the GPR 17 isoform tested some functional activity of uracil nucleotides was seen, although only at low, or high micromolar concentrations, respectively.
WO 2005/103291 suggested the endogenous molecules 5 amino levulinic acid (5-ALA) and porphobilinogen (PBG) as activating ligands for GPR17, and a screening assay using these GPR 17 agonists. However, the reported affinity of 5-ALA and PBG is quite low and the amounts needed in the assays are significant, namely in the three digit micromolar range for 5-ALA or even in the mM range for PBG, which make both compounds not well suited for use in routine screening assays or even high throughput screenings. Moreover, PBG is a chemical unstable, reactive compound which rapidly decomposes after exposure to air and light, making it impractical to handle on a routine basis.
Accordingly, a need exists for the identification of improved GPR 17 agonists, which can be used as an easy and cheep but robust and reliable tool for the identification of GPR17 antagonists in various experimental settings.
3-(2-Carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid and some analogs had been previously described as allosteric inhibitors of fructose-1,6-biphosphatase (Wright et al, MBCL 2003, 13, 2055), and as antagonists of the NMDA receptor associated glycine binding site (Salituro, J Med Chem, 1992, 35, 1791; U.S. Pat. No. 4,960,786). However, these compounds have not yet been described as GPR17 modulators.